Analysis of SLC22A18 promoter methylation and protein expression in GBM cell cultures explanted from surgical specimens. (A) CpG island of SLC22A18 gene with location and sequence of methylation-specific polymerase chain reaction (MSP) primers (uf, forward U-MSP primer; ur, reverse U-MSP primer; mf, forward M-MSP primer; mr, reverse M-MSP primer). (B) Representative images of MSP analysis of SLC22A18 promoter methylation. Amplification products with specific primers for unmethylated (u) and methylated (m) DNA sequences are indicated, and scoring as unmethylated (u), methylated (m), and mixed (u/m) is shown. Positive controls for unmethylated (pcu) and methylated (pcm) sequences were included. M, size marker. (C) Representative images of western blotting analysis of SLC22A18 protein expression, and levels of relative expression were gained by the densitometric analysis of Western blotting compared with the SLC22A18 expressing human neurons included as positive control (pc) and set arbitrarily as 1. β-actin was used as loading control. (D) Scatter gram analysis of SLC22A18 protein expression in two subgroups with methylated and unmethylated SLC22A18 promoter sequences.